This project aims to characterize an overlapping bacterial quorum sensing (QS) system between Streptococcus pyogenes (Group A Streptococcus, GAS) and Streptococcus agalactiae (Group B Streptococcus, GBS) and the role of this system in biofilm formation and virulence. Both GAS and GBS are important human pathogens causing a variety of diseases, often biofilm related, and leading to significant human morbidity and mortality. Understanding communication between them will allow development of novel QS inhibitors to modulate virulence of both organisms. In GAS, the QS signaling peptides Shp2 and Shp3 enhance biofilm formation. These Shp peptides are regulated by two Rgg regulators, Rgg2/Rgg3 which, in turn, are regulated by the Shp peptides. In GBS, a small hydrophobic peptide (shp) almost identical to Shp2 has been found directly adjacent to a homolog almost identical to Rgg2 termed RovS. RovS has been shown to be a regulator of a multitude of virulence genes in GBS. Our preliminary data demonstrate that the production of the Shp by GBS can activate Shp2/3 regulated genes in GAS signifying interspecies communication between GAS and GBS via this QS system. The observed role of these QS systems in biofilm formation and virulence as well as their ability to mediate cross-species signaling clearly demonstrates the importance of understanding their mechanism of action. The results of these experiments may provide novel targets for QS inhibitors to prevent biofilm formation and decrease morbidity and mortality associated with streptococci infections. To examine the role of QS and interspecies communication between GAS and GBS on virulence and biofilm formation, we will create strains of GBS without RovS and/or the Shp peptide and use both luciferase reporters and qRT-PCR to measure gene expression in knockout strains. We will also make gene replacement strains swapping Rgg2 and RovS between GAS and GBS to determine whether these proteins can complement each other. Because Shp2/3 have been shown to enhance biofilm formation in GAS, the effects that each mutant has in biofilm development will be tested. To determine how peptide expression overlaps with biofilm formation, we will use RNASeq to determine the transcriptome profiles of GAS and GBS growing planktonically or in biofilms, and with or without the presence of the Shp peptide. Finally, we will test whether deletion of these genes alters the virulence of both GAS and GBS in mouse models of infection and carriage. Taken together, these studies will provide great insight into the ability of two important human pathogens to communicate with each other in clinically relevant settings. The data from mutational studies will allow us to characterize the first signaling system that allows communication between different species of streptococci.